晋南牛SREBP1基因调控前体脂肪细胞分化的研究

doi:10.11843/j.issn.0366-6964.2024.11.019

摘要/Abstract

摘要：

旨在探究SREBP1基因对晋南牛前体脂肪细胞分化的影响，并通过RNA-Seq技术探究该基因在晋南牛前体脂肪细胞分化过程中的调控机制。本研究利用SREBP1基因的过表达载体和siRNA转染晋南牛前体脂肪细胞，油酸诱导分化后采用油红O染色观察脂滴累积情况，检测甘油三酯(triglyceride，TG)含量以探究SREBP1基因对其分化的影响。利用RT-qPCR和蛋白免疫印迹技术(Western blotting，WB)检测过表达SREBP1基因的前体脂肪细胞中相关基因mRNA和蛋白水平变化。RNA-Seq检测干扰SREBP1基因的脂肪细胞，筛选差异表达基因(differentially expressed genes，DEGs)进行KEGG通路富集分析，并对差异表达基因进行验证，以探究SREBP1基因参与调控晋南牛前体脂肪细胞分化相关的潜在靶基因。每个处理均设置3个重复。结果表明：1)过表达SREBP1基因能够促进前体脂肪细胞内脂滴的累积、极显著提高细胞内TG含量(P < 0.01)，干扰SREBP1基因则会抑制脂滴形成。2)过表达SREBP1基因后FABP4的mRNA表达量显著升高(P < 0.05)，FABP7、LPL的表达量极显著升高(P < 0.01)，WB结果显示FABP4蛋白表达水平明显升高；3) RNA-Seq共筛选到227个DEGs，其中包括67个上调基因和160个下调基因。京都基因和基因组百科全书(KEGG)富集分析结果表明差异基因富集到PPAR信号通路和不饱和脂肪酸生物合成等相关通路，干扰SREBP1基因后，差异表达基因FABP4、LPL和FABP7的mRNA表达量均极显著降低(P < 0.01)，FABP4的蛋白表达水平也明显降低。由此可知，SREBP1基因对于晋南牛前体脂肪细胞分化具有促进作用，过表达该基因可促进细胞内TG累积，并且极有可能是通过对与脂质分化相关的PPAR信号通路和不饱和脂肪酸生物合成通路的调控实现的。本研究结果为探究SREBP1基因在晋南牛前体脂肪细胞分化过程中的调控机制提供理论参考。

关键词: 前体脂肪细胞, SREBP1, 诱导分化, RNA-Seq, PPAR

Abstract:

This study aimed to investigate the effect of the SREBP1 gene on the differentiation of Jinnan cattle precursor adipocytes and to explore the regulatory mechanism of this gene in the differentiation of Jinnan cattle precursor adipocytes by RNA-Seq technology. We transfected Jinnan cattle preadipocytes with an overexpression plasmid of SREBP1 gene and siRNA, and after differentiation induced by oleic acid, oil red O staining was used to observe the accumulation of lipid droplets, and triglyceride (TG) content was tested to explore the influence of SREBP1 gene on their differentiation. RT-qPCR and Western blotting (WB) were used to detect changes at mRNA and protein levels of related genes in preadipocytes overexpressing the SREBP1 gene. RNA-Seq was used to detect adipocytes that interfered with the SREBP1 gene, screened differentially expressed genes (DEGs), performed enrichment analysis of the KEGG pathway, and verified the differentially expressed genes. The potential target genes of the SREBP1 gene involved in regulating the differentiation of precursor adipocytes of Jinnan cattle were explored. Three replicates were set up for each treatment. The results showed as follows: 1) Compared with the control group, overexpression of SREBP1 gene could promote the accumulation of lipid droplets in precursor adipocytes and significantly increase the intracellular triglyceride content (P < 0.01), knockdown of SREBP1 gene could inhibit lipid droplet formation. 2) FABP4 mRNA expression significantly increased after overexpression of the SREBP1 gene (P < 0.05), the expression of FABP7 and LPL were significantly increased (P < 0.01), WB results showed that FABP4 protein expression level increased obviously; 3) A total of 227 DEGs were detected by RNA-Seq, including 67 up-regulated genes and 160 down-regulated genes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that differentially expressed genes were enriched into PPAR signaling pathway and unsaturated fatty acid biosynthesis pathway, the mRNA expression of differentially expressed genes FABP4, LPL, and FABP7 decreased significantly after interfering with SREBP1 gene (P < 0.01), the protein expression level of FABP4 also decreased obviously. In conclusion, the SREBP1 gene can promote the differentiation of precursor adipocytes of Jinnan cattle, and overexpression of this gene can promote intracellular TG accumulation, which is most likely achieved through the regulation of the PPAR signaling pathway and unsaturated fatty acid biosynthesis pathway related to lipid differentiation. The results of this study provide a theoretical reference for exploring the regulatory mechanism of the SREBP1 gene in the differentiation of bovine precursor adipocytes.

Key words: precursor adipocytes, SREBP1, induced differentiation, RNA-Seq, PPAR

中图分类号:

S823.2

张唯玉, 程景, 许家宝, 王静, 陶薪燕, 李博, 张亚伟, 张丹丹, 张宁, 郝振凯, 周琛帛, 张元庆. 晋南牛SREBP1基因调控前体脂肪细胞分化的研究[J]. 畜牧兽医学报, 2024, 55(11): 5003-5017.

Weiyu ZHANG, Jing CHENG, Jiabao XU, Jing WANG, Xinyan TAO, Bo LI, Yawei ZHANG, Dandan ZHANG, Ning ZHANG, Zhenkai HAO, Chenbo ZHOU, Yuanqing ZHANG. Regulation of Preadipocyte Differentiation by SREBP1 Gene in Jinnan Cattle[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5003-5017.

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基因Gene	登录号GenBank No.	引物序列(5′→3′) Primer sequence	产物长度/bp Product size
固醇调节元件结合蛋白1 SREBP1	NM_001113302.1	F：CGACACCACCAGCATCAACCACG	119
		R：GCAGCCCATTCATCAGCCAGACC
β肌动蛋白β-actin	AY141970.1	F：GCACCACACCTTCTACAACGAGC	179
		R：CAGAGGCATACAGGGACAGCAC
脂蛋白脂肪酶LPL	NM_001075120.1	F：GCCGCAGACAGGATTACAGG	107
		R：CCAGGAATGAGGTGGCAAGT
脂肪酸结合蛋白4 FABP4	NM_174314.2	F：TGAGATTTCCTTCAAATTGGG	202
		R：TGAGATTTCCTTCAAATTGGG
脂肪酸结合蛋白7 FABP7	NM_001078162.2	F: GCTTGGTGTGGGCTTTGCC	154
		R: GGTTTCATCAAACTCTTCTCCCAG

引物名称Primer name	引物序列(5′→3′)Primer sequence
si-365-sense	CCUGAAGAUAUACCCAUCUTT
si-365-antisense	AGAUGGGUAUAUCUUCAGGTT
si-1051-sense	GCUCUUCCAUCAAUGACAATT
si-1051-antisense	UUGUCAUUGAUGGAAGAGCTT
si-3142-sense	GGAGGGUAUUCCUACAUGATT
si-3142-antisense	UCAUGUAGGAAUACCCUCCTT
si-con-sense	UUCUCCGAACGUGUCACGUTT
si-con-antisense	ACGUGACACGUUCGGAGAATT
si-con-FAM-sense	UUCUCCGAACGUGUCACGUTT
si-con-FAM-antisense	ACGUGACACGUUCGGAGAATT

基因Gene	差异倍数log₂FC
SREBP1	-2.436 97
FABP4	1.616 90
FABP7	-1.032 92
PLTP	2.365 92
FADS2	-2.587 32
LPL	-1.114 32

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